Preservation of early Tonian macroalgal fossils from the Dolores Creek Formation, Yukon

The rise of eukaryotic macroalgae in the late Mesoproterozoic to early Neoproterozoic was a critical development in Earth’s history that triggered dramatic changes in biogeochemical cycles and benthic habitats, ultimately resulting in ecosystems habitable to animals. However, evidence of the diversification and expansion of macroalgae is limited by a biased fossil record. Non-mineralizing organisms are rarely preserved, occurring only in exceptional environments that favor fossilization. Investigating the taphonomy of well-preserved macroalgae will aid in identifying these target environments, allowing ecological trends to be disentangled from taphonomic overprints. Here we describe the taphonomy of macroalgal fossils from the Tonian Dolores Creek Formation (ca. 950 Ma) of northwestern Canada (Yukon Territory) that preserves cm-scale macroalgae. Analytical microscopy, including scanning electron microscopy and tomographic x-ray microscopy, was used to investigate fossil preservation, which was the result of a combination of pyritization and aluminosilicification, similar to accessory mineralization observed in Paleozoic Burgess Shale-type fossils. These new Neoproterozoic fossils help to bridge a gap in the fossil record of early algae, offer a link between the fossil and molecular record, and provide new insights into evolution during the Tonian Period, when many eukaryotic lineages are predicted to have diversified.


Results
Field sampling was conducted in 2018 and consists of 90 collected slabs and 339 observed fossil specimens. A range of preservational fidelity has been identified, which we have scored into three qualitative taphonomic grades: Grade 1, Well-preserved specimens (e.g., HCS-59, HCS-72; Fig. 2) that include a clearly defined uniseriate and filamentous cellular organization; Grade 2, moderately preserved specimens (e.g., HCS-25, HCS-40; Fig. 3) showing identifiable cellular boundaries (cross walls) and lateral cell walls (side walls) for most of the thallus; or Grade 3, poorly preserved specimens (e.g., HCS-23, HCS-44; Fig. 4), with infrequent preservation of cell walls. All elemental data herein is reported in mean normalized weight percent, wt%.
Grade 2: moderately preserved specimens. Moderately preserved specimens (grade 2) were difficult to observe in backscattered SEM imaging owing to compositional similarity to the host rock matrix or dominance of thin carbonaceous material, though their cellular arrangement was still discernable (Fig. 3). Specifically, correlative imaging with light photography and ATLAS SEM mosaics was the clearest way to observe the fossil morphology (Fig. 3a). These fossils resemble carbonaceous compressions (e.g., HCS-25, HCS-40). They typically preserve a highly reflective black (or grey) surface similar to the appearance of other carbonaceous  35,37 . The grade 2 specimens contain greater concentrations of aluminium (Fig. 3j,p,y) when compared to the grade 1 and 3 fossils, however, Al is not always enriched in the fossil when compared to the matrix. For example, HCS25-5 (fossil = 15.81% vs. matrix = 11.80%) and HCS25-1 fossils (fossil = 15.67% vs. matrix = 12.51%) are enriched in aluminium compared to the matrix while HCS40-1 shows the opposite trend (fossil = 9.71% vs. matrix = 16.53%, Fig. 3v,y). Grade 2 fossils show subtle enrichment of potassium in HCS25-5 (fossil = 6.71% vs. matrix = 4.69%) and HCS25-1 fossils (fossil = 8.06% vs. matrix = 3.87%, Fig. 3k,q,y) with limited carbon enrichment. The HCS-40 fossil is not enriched with potassium (Fig. 3w,y) but features a pronounced iron enrichment compared to the matrix (fossil = 10.42% vs. matrix = 0.00% [not detected], Fig. 3t,y) and other grade 2 specimens (iron in HCS25-5 fossil = 2.66%; HCS25-1 fossil = 2.54%, Fig. 3h,n,y). Pits and platy linear features ( Fig. 3b-d, see Fig. 3y for "framboids" and "linear features") were observed throughout the grade 2 fossils; platy features have increased concentrations of iron, potassium, and aluminum ( Fig. 3b,j,k,p,q,y), while the pits have framboidal to cubic structures with increased iron relative to the matrix (Fig. 3d,h,n,y).   (Fig. 4). When compared with taphonomic grade 2, there is only a slight increase in iron enrichment relative to the host-rock matrix (Fig. 4h,n,s "matrix" vs "fossil"), while taphonomic grade 1 retains higher iron. Interestingly, some of the poorly preserved specimens show localized iron along the external boundary and slight elevation within the organism (Fig. 4f,n), while others have a more noticeable increase in iron concentrated along the side wall (Fig. 4c,h). The lack of cellular detail observed in grade 3 specimens starkly contrasts with the cellular organization highlighted by iron within the filamentous thallus in grade 1 specimens. Most grade 3 specimens demonstrate uniformly distributed iron throughout the fossil, obscuring cell boundaries, although the overall ribbon-shape remains clear. www.nature.com/scientificreports/ Interpretation of taphonomic grades. The differences between the taphonomic grades should be interpreted to represent the quality of morphological characters preserved by distinct processes that may, or may not, be chemically or mineralogically related to each other. The grades therefore represent overall taphonomic fidelity-from well-preserved (grade 1), to moderately preserved (grade 2), to poorly preserved (grade 3)-but do not necessarily represent a continuum of preservation or later alteration from one grade to the next. Preservational modes, on the other hand, represent the composition of the fossil as preserved, including influences of pyritization and aluminosilicification as noted above. Specimens representing different taphonomic grades and preservational modes were recovered from the same bedding planes, with some individual fossil specimens showing variation in both grade and mode. Nevertheless, we can draw some general inferences from our observations that relate grade and mode. Grade 1 specimens preserve detailed morphology and contain the highest concentrations of carbon, and relatively higher concentrations of iron, aluminum, and magnesium when compared to the matrix. Grade 2 specimens are enriched in iron and carbon as compared to grade 1, but, of the three taphonomic grades presented, they are comparatively most similar to the matrix, perhaps owing to the common preservation via aluminosilicification. Grade 3 specimens are poorly preserved, and usually show the high iron concentrations and only a slight elevation of carbon. Although we cannot rule out the possibility that "grade 3" specimens represent a different species, this is unlikely based on the consistent morphology including a filamentous structure with uniform thallus and cell widths throughout the population regardless of grade.

Discussion
Preservation of organic material can involve several mineralization processes 8,9 . Non-mineralized macroalgae are commonly preserved as carbonaceous compressions 1,38,39 . The outward appearance and mineralogical composition of the Dolores Creek fossils suggests that they were preserved under circumstances comparable to other Neoproterozoic macroscopic carbonaceous compressions, such as the Chuaria-Tawuia assemblage in the Little Dal Group 40,41 , and are otherwise generally analogous to broader BST fossil preservation 10,[42][43][44] . The carbonaceous compressions that preserve the tubular metazoans of the Ediacaran Gaojiashan biota are also associated with pyritization and aluminosilicification 8,9,45 . These kerogenized remains are composed of recalcitrant aliphatic polymer chains resulting from the polymerization of the original organic matter 46 , but preservation can be gradational and also involve other integrated taphonomic pathways (e.g., pyritization, aluminosilicification) 8,47 . The Ediacaran Miaohe and Lantian biotas, for example, are preserved as carbonaceous compressions associated with densely packed framboidal pyrite 48 . The Dolores Creek macrofossils retain little carbonaceous material, and they are broadly preserved as aluminosilicate-templated compressions with associated iron oxides www.nature.com/scientificreports/ from presumed oxidative weathering of pyrite 8,36,44,49 . The observable range in taphonomic quality appears to vary with their preservational composition: the less well-preserved specimens (grade 3) tend to be preserved primarily by iron oxide coatings, whereas higher-fidelity specimens (grades 1-2) instead have consistent clay mineral coatings, with iron oxides limited to the recalcitrant cell walls (Figs. 2, 3, 4). This pattern further applies to the shiny dark grey to black clay veneers, which resemble organic carbon but are composed of aluminum, magnesium, and iron in well-preserved grade 1 specimens, as opposed to potassium and iron in moderately preserved grade 2 specimens. Textures within the fossils include micrometric linear features composed mostly of iron and aluminum platy clay mineral textures (Fig. 3d). These linear features are approximately 2 μm in width, substantially thinner than the width of longitudinal striations (~ 100 μm; see Fig. 2A,F of 28 ), which were interpreted as ribbed cell wall ornamentation 28 . High densities of circular pits are interpreted to represent external molds of pyrite framboids that have been removed physically (plucked-out from slab splitting) or chemically (dissolution) (Fig. 3c,d), although chemical weathering can sometimes result in iron oxide pseudomorphs after pyrite 37,50 . Comparable filamentous fossils were observed throughout the host rock samples that have split along bedding planes, confirming the high-density features observed during µCT analysis (Fig. 5).
The preservation of the Dolores Creek macrofossils is unusual as they have relatively low carbon enrichment compared to what would typically be observed in carbonaceous compression-type fossils 49,51 . This mode of preservation involves the adsorption of ferrous iron (Fe 2+ ) or aluminum by the labile tissues, which inhibits bacterial decay, allowing enough time for the original organic matter to mature to kerogens by the loss of volatile components 44,52 . Structural polysaccharides like cellulose, a common constituent of algal cell walls 53 , are likely to adsorb positively charged cations such as ferrous iron, which in turn protects them from enzymatic hydrolysis 52 www.nature.com/scientificreports/ thereby interfering with their impending breakdown 54 . Ferrous iron would be readily available if the organism is buried in sediments beneath anoxic water masses, or if the resulting oxidation of organic matter by iron (III) reducing bacteria led to ferruginous pore waters. Importantly, carbon can be replaced or templated during early diagenetic mineralization, including aluminosilicification and pyritization. In the Dolores Creek macrofossils, in addition to the relative absence of carbon, there is evidence of iron enrichment on the fossil surfaces interpreted as clays and weathered pyrites, with slightly more iron on the cell walls relative to other parts of the fossil. This pattern is similar to that seen in some fossils from the Cambrian Chengjiang biota, which are also deficient in organic carbon and preserved by pyritization 37 . It is possible that the initial kerogenization of the fossils was followed by replacement by iron rich-minerals (iron oxides/hydroxides, pyrite) 34 or that the fossils experienced extensive post-burial loss of organic carbon. Given that the fossils contain very little to no carbon, the specimens likely experienced extensive degradation and the fossils were templated by clays followed by the loss of organic material. Clays play an important role in the protection of the labile tissues from degradation during early diagenesis 33,34,36,51,55 . It has been suggested that the clay associated with BST fossilization may have resulted from later stage metamorphism 35,56,57 ; however, this is an unlikely explanation for the Dolores Creek specimens as they have only undergone, at most, up to zeolite-grade metamorphism (less than 250 °C and low pressure) 58 . On the other hand, several proposed taphonomic mechanisms invoke the role of clays in close association with the decaying carcass, whether attachment of existing clays in the environment or precipitation of clays de novo. Clays have been demonstrated to inhibit decay 33,55 and effectively replicate labile tissues (i.e., aluminosilicification) 42,44,51 . Taphonomic experiments further demonstrate the plausibility of authigenic clay formation during (and facilitated by) organic degradation [59][60][61] . However, it is important to note that experimental studies have also shown that some reactive clays, like montmorillonite, can instead have a deleterious effect on preservation 47 . Based on the observed mineral composition in the Dolores Creek fossils, aluminum-or iron-rich clays were likely essential to their fossilization 55 . Additionally, smectite clays 62 were likely involved in the preservation of grade 1 fossils, while potassium-rich illite potentially aided in preservation of the grade 2 fossils, which suggests a possible alteration product of the original Al-rich clays or heterogeneity in the paleoenvironment. Aluminum-rich kaolinite is preferentially preserved in BST fossils following metamorphic alteration, supporting the hypothesis that early diagenetic interactions with the organism protected the clay from metamorphic transformation 44 . Regardless, kaolinite can be altered to the iron-rich clay berthierine during diagenesis when Fe 2+ is present in the pore waters 36 . The alteration of kaolinite can explain the limited aluminum enrichment yet abundant iron enrichment in the Dolores Creek macrofossils.
Pyritization also contributes to the iron enrichment observed in the fossils and specifically requires a source of organic material (i.e., the organism) buried within an environment rich with iron and sulfate 18,63 . Sulfate is converted to bisulfide (HS -) when sulfate-reducing bacteria oxidize organic material under normal seawater pH conditions. Details of soft tissues can be lost by the overproduction of pyrite; to ensure fossilization, pyritization must be reasonably focused on the organisms being fossilized while sulfate reduction in the surrounding matrix is supressed. Such conditions are hypothesized to be a result of limited organic availability outside of the fossil materials or otherwise low TOC levels 19,20,64 . If the process is inhibited by limited organic matter, then recalcitrant tissues can be preserved by authigenic pyrite, while cellular level details are lost during degradation and subsequent diagenesis 18,63 . To account for the exceptional preservation observed in the Dolores Creek Formation, we infer that pyritization was likely restricted early in the taphonomic process due to limited availability of sulfate 65 . This scenario contrasts with other examples (e.g., the Ediacaran Gaojiashan Lagerstätte of South China) where pyritization was unimpeded by limited sulfate or reduced iron 8,9,63 , consistent with relatively high levels of marine sulfate at this time 66 . Taphonomic grades 1 and 2 are inferred to have experienced minimal pyritization, allowing the cellular structures to be preserved in detail (except in the rare cases of 3D preservation, see below). These well-to-moderately preserved specimens were likely exposed to sulfate ions after the cellular structures were protected by aluminosilicates or stabilized as kerogen. Poorly preserved grade 3 specimens with no cellular structures potentially lacked protective clay templates 44 , based on the pervasive pyritization observed, and suffered degradation by sulfate-reducing bacteria in the presence of an adequate supply of sulfate ions. Pyritization is known to aid in the three-dimensional preservation of metazoan-grade soft tissues and refractory tissues of plants 34,63,67 , but the three-dimensional preservation observed in the Dolores Creek macrofossils illustrates that focused pyritization on cell walls can accomplish comparable taphonomic fidelity in macroalgae. A similar pattern was identified in fossil plants from the Eocene London Clay: although parenchymatous cell walls were coalified, the recalcitrant (lignified) cell walls were instead pyritized 68 . Proterozoic fossil specimens of Grypania and Vendotaenia can also be preserved by pyrite crystals in iron-rich clays and other aluminosilicate minerals [69][70][71] and may share similar taphonomic pathways with the Dolores Creek fossils. The 3D preservation observed in the rare examples of Dolores Creek macroalgae appears to have been aided by the thin exterior veneering of pyrite, but the overwhelming majority of fossils are preserved as flattened specimens, indicating microenvironmental and/or taphonomic heterogeneities.
The sedimentary facies that host non-calcified macroalgae fossils in the Proterozoic are broadly similar, with deposition in shallow subtidal marine environments influenced by episodic sedimentation events 72 . Such rapid burial is essential in exceptional preservation through carbonaceous compression or related taphonomic pathways 9,73,74 . These events can transport the organisms into low oxygen settings or bury them beneath enough sediment to restrict diffusion of oxidants from overlying seawater. In addition, high sedimentation rates in settings influenced by gravity flows can dilute the sedimentary organic carbon, which is important because high contents of total organic carbon correlate with decreased preservation of Proterozoic organic walled microfossils 75 . These rapid sedimentation events are also taphonomically advantageous because they impede degradation by efficient aerobic microbes. The Dolores Creek macroalgae occur in upper slope facies, but the organisms were likely derived from the shelf margin, which was likely in the photic zone based on the abundance of stromatolites.  9 , the documented range of preservation seen in the Dolores Creek fossils is hypothesized to result from a dynamic and heterogeneous environment where the availability of organic material and sulfate dictated the extent to which the specimens were pyritized (Fig. 6). We hypothesize that grade 1 specimens have clear cross walls, striae and rare 3D specimens preserved by pyrite (later oxidized to iron oxides) and iron-rich clays (Fig. 6e), although aluminum-rich clays are also implied to have been involved in early preservation before subsequent diagenetic alteration. Cross-walls are also visible in grade 2 specimens as ellipses or bands preserved by clays (likely potassium-rich clays) (Fig. 6f). Grade 3 specimens are poorly preserved by pyrite that was later oxidized to iron oxides (e.g., limonite) with cross-walls being exceedingly rare (Fig. 6g). Based on these observations, we propose the following steps to account for the exceptionally preserved fossils: (1) organisms were transported downslope by gravity flows and rapidly buried (Fig. 6a); (2) algae then experienced decay and compaction, compressing the organisms into a 2D form (with some rare 3D specimens preserved, Fig. 6e); (3) clay templating and pyritization variably preserves the fossils (Fig. 6b-g).
Biases in the fossil record can obscure the evidence of important biological events, which are crucial to the evaluation of ancient life and their paleoenvironments. The Dolores Creek macrofossils were interpreted as www.nature.com/scientificreports/ green algae based on their large size, putative holdfasts, and cell wall ornamentation 28 . However, many of these characteristics are absent from the moderately and poorly preserved fossils, making it impossible to differentiate between a cyanobacterial or algal origin in the poorly and moderately preserved fossils. Thus, exceptionally preserved fossils are required to accurately document the significant biological innovations among eukaryotes, including their origins 76,77 , acquisition of plastids 78 , advent of multicellularity 79 , and the onset of eukaryovory 80 . Rapid burial due to gravity flows, the availability and/or limitation of sulfate and clays, and the inhibition of decay all contribute to the exceptional preservation observed. These factors would only occur in specific depositional environments and at specific times, for example in marine, shelf margin settings with sufficient slope and sediment input to result in periodic gravity flows at a time of relatively low marine sulfate concentrations to limit pyritization. Clay formation and alteration are also influenced by the tectonic setting of the depositional environment; detrital clay deposition is favored in low energy environments, and the prevalent clay mineralogy is dictated by sediment provenance. These environmental controls are supported by our observations of the Dolores Creek Formation where macroalgal fossils were recovered from down slope debrites, whereas fossils have yet to be recovered from the stromatolitic bioherm intervals that the algae likely inhabited 28 . While this sedimentological relationship between the source of the original organisms and their site of burial and preservation highlights the likely rarity of exceptional fossils, it also provides a useful target for depositional facies that are more prone to their preservation. Macroalgae from the Tonian Dolores Creek Formation are variably preserved by pyritization and clay templating, similar to other carbonaceous compressions from the Proterozoic, although with generally low remaining carbon content. The early templating by clays and pyrite followed rapid burial and facilitated exceptional preservation. Grade 1 organisms have the best-preserved morphology, which is critical in identifying their macroalgal affinities. Grade 2 and 3 fossils experienced greater diagenetic alteration, with grade 3 fossils having been extensively degraded by sulfate reducing bacteria in an environment where sulfate was available but not replete. Based on our results, future fossil searches should target silty shales along shelf margin to fore-slope paleoenvironments, where organic remains would be rapidly buried during episodic gravity flow events. Lagerstätten are necessary to preserve cellular level structures, which supports the hypothesis that the documented distribution of Proterozoic eukaryotic fossils is sketchy and taphonomically biased. Continued work on Tonian strata of northwestern Canada will undoubtedly contribute to further understanding of exceptional fossil preservation and the role macroalgae played in Tonian ecosystems.

Methods
Material and preparation. Slabs containing fossil specimens were recovered from the Dolores Creek Formation of the Mackenzie Mountains Supergroup where it outcrops near the headwaters of Hematite Creek in the Wernecke Mountains (Fig. 1). Seven in-situ beds were targeted for sampling after observing fossils in float material. Each slab contains macroalgal specimens, but the number of fossils per slab ranges from 1 to 100 s, sparsely to densely packed. Individual fossils from the in-situ beds display a range of preservation, from exceptional preservation with cellular-level details to poor preservation with only gross morphology (Figs. 2, 3, 4). All fossils analysed herein are reposited at the Royal Ontario Museum (Toronto, Ontario, Canada) and the Yukon Geological Survey (Whitehorse, Yukon, Canada). Fossils representing a range in preservation across the qualitative taphonomic grades were selected for further analyses using scanning electron microscopy (SEM), energydispersive x-ray spectroscopy (EDS), and tomographic X-ray microscopy (µCT).

Analytical methods.
To investigate the mineralogical differences observed in the macroalgal fossils, sample slabs exemplifying the range of taphonomic scores were selected for analysis at the University of Missouri X-ray Microanalysis Core using a Zeiss Sigma 500 variable-pressure, field emission scanning electron microscope (SEM) equipped with dual, co-planar Bruker XFlash energy dispersive X-ray spectrometers (EDS). Identical beam and chamber conditions were used for SEM imaging and EDS analyses: 20 keV beam accelerating voltage, 40 nA current, beam apertures of 60 µm (imaging) and 120 µm (EDS), a working distance of 16 mm (± 0.2 mm; flat samples allowed for minimal variation), and 20 Pa chamber pressure with a 99.999% nitrogen atmosphere. The larger aperture selection for EDS analyses serves to improve X-ray count rate, which was greater than 100 kilocounts per second in all maps and point analyses. Spatial distribution of elemental composition was determined using EDS elemental mapping (360 s live-time), supplemented with point spectral collection (60 s per point, n = 150 points over 6 slabs including both fossil and host rock points). Both spectrometers were used in tandem to help mitigate any topographic artifacts, which were likely minimal given the flat nature of the majority of the slabs and specimens. In addition, maintenance of equivalent operating conditions across all samples helps to minimize between-sample variation. EDS point data are reported in normalized weight percentages in Figs. 2, 3, 4 and in the Supplementary Materials. A high-definition 5-segment backscatter detector and a cascade current detector were used, respectively, to conduct Z-contrast backscattered (BSE) and low-vacuum secondary electron (SE) imaging. Large-area SEM mosaic images were conducted and compiled using the ATLAS workflow (Fibics, Inc.). One exceptional specimen that preserves 3D morphology (HCS-W18-72; Fig. 2) was further investigated via µCT volume imaging using a Zeiss Xradia 510 Versa. Operating conditions are as follow: 80 kV source voltage, 7 W source power, LE3 filter, 0.4 × objective, 4.5 s exposure, 2001 projections at 360 degrees, and a voxel size = 11.09 μm.